Alga strain

ABSTRACT

An alga named Dunaliella bardawil is similar to D. salina except that its length is 15-19 μm, its diameter 10-14 μm and its volume 200-1000 μm 3 , and it contains large quantities of carotene and glycerol.

This application is a continuation-in-part of utility application, Ser.No. 918,802, filed June 26, 1978, the contents of which are herebyincorporated by reference.

The present invention relates to a new and distinct variety of algaknown as Dunaliella bardawil (ATCC 30861). This new species, which hasbeen isolated and cultivated as a pure algal culture, belongs to theClass of Chlorophycae, Order of Volvocales and has been deposited in theUniversity of Texas International Culture Collection of Algae, Austin,Tex., 78712.

The algae of the present invention are unicellular motile cells of about15 to 19 μm long and 10 to 14 μm in diameter, which are broadly ovoid orellipsoid in shape with a fine elastic periplast but with no rigid cellwall. Two flagella 1.5 to 2 times the length of the cell emerge from oneedge of the long cell axis, one large chloroplast occupies about halfthe cell volume and is often arranged in a cup-shape around the nucleus.A single median pyrenoid is embedded in the basal portion of thechloroplast and surrounded by starch granules. The cell volume is 200 to1000 μm³. The cells reproduce vegetatively by longitudinal division ofthe motile cells.

For maximal β-carotene production the algae must be cultivated under anadequately high intensity of illumination, and this is best carried outoutdoors in sunlight. If artificial light is used, the intensity ofillumination ought to be at least about 1500 f.c. When cultivatedoutdoors the depth of the water ought not to exceed about 15 cm, and theoptimum is about 5 cm.

In the following the content of glycerol, carotene, chlorophyll andprotein of the algae are specified, both when grown outdoors, and underlow intensity (200 f.c.) artificial illumination.

    ______________________________________                                        Content of β -carotene, Glycerol, Chlorophyll and Protein                under favorable conditions:                                                                Growth outdoors,                                                                         Growth indoors,                                                    3 M NaCl   3 M NaCl                                              ______________________________________                                        β -carotene pg/cell                                                                     50-90         8-16                                             Glycerol pg/cell                                                                             300-400      200-275                                           Chlorophyll pg/cell                                                                          4-8          10-16                                             Carotene mg/g dry wt.                                                                        50-90        10-21                                             Glycerol mg/g dry wt.                                                                        300-400      270-370                                           Chlorophyll mg/g dry wt.                                                                      8-12        13-21                                             Protein mg/g dry wt.                                                                         300-400      300-400                                           ______________________________________                                    

The cultivation is carried out either in an artificial medium or onseawater adjusted so as to contain the required nutrients and saltconcentration.

An artificial medium ought to contain the following nutrients:

NaCl, 1-5 M, and preferably 3-4 M; Mg⁺⁺, 1-500 mM, and preferably 5 mM;K⁺, 1-10 mM; Ca⁺⁺, 0.1-20 mM and preferably 0.2-0.4 mM, iron source,such as Fe-EDTA 0.5-45 μM, and preferably 1-2 μM; SO₄ ⁻⁻, 1-5 mM;Nitrogen source, as NO₃ ⁻, 1-20 mM and preferably 3-4 mM, or NH₄ NO₃,0.5-2.5 mM and preferably 1-2 mM; phosphate, 0.01-1 mM.

It is possible to use seawater augmented by addition of variousconstituents or concentrated by partial evaporation and addition ofcertain constituents. This ought to have a sodium chloride content of upto 1-4.5 M NaCl and preferably 3 M NaCl supplemented with a nitrogensource such as NH₄ NO₃, 0.5-2.5 mM, and preferably 1-2 mM, phosphatesource such as KH₂ PO₄, 0.01-1 mM, and preferably 0.1 mM, and an ironsource such as FeCl₃ -EDTA, 0.5-50 μM, preferably 2 μM.

There must be provided a suitable and adequate source of carbon such as10 mM NaHCO₃ or CO₂ at about 300 liter CO₂ per m³ growth medium per day.When CO₂ is used as the carbon source, it is added by demand via a pHcontrolled solenoid valve. Other inorganic carbon sources such as CaCO₃,organic carbon sources such as sewage water, etc., are also suitable forgrowth.

The optimum pH for cultivation is between 7.0 and pH 9.0 and this isadvantageously adjusted by adding required quantities of carbon dioxide,mineral acids such as hydrochloric acid or nitric acid, via a pHcontrolled valve.

The optimum temperature of cultivation is in the range of about 25°-35°C., and the algae withstand temperatures of about 4° C. to 40° C.

Various contaminant microorganisms, such as fungi, zooplankton,crustaceae etc. constitute a problem under conditions of large-scalecultivation and this can be overcome to a very large extent by carryingout the cultivation at a sodium chloride concentration of above about3.0 M. The content of glycerol increases with the concentration of thesodium chloride in the growth medium. It is desirable to effectcultivation around 3.0 M NaCl.

In order to obtain a high content of carotene it is necessary to provideadequate intensity of illumination, as pointed out above. It is furtherhelpful to supply a limiting concentration of nitrogen which can beprovided in forms such as potassium nitrate, sodium nitrate, ammoniumnitrate or by ammonia. When cultivation is carried out at a high sodiumchloride content and under strong illumination, the nitrogen contentought to be below 4 mM; when cultivation is effected under lesserintensitites of illumination and at a smaller sodium chlorideconcentration, the nitrogen content ought not to exceed about 1 mM foroptimum carotene content. A diurnal cycle ought to be maintained asunder constant intense illumination severe inhibition of growth takesplace.

The rate of reproduction of the algae is higher when the concentrationof sodium chloride is not too high. The rate at about 1.5 M is abouttwice that at 4 M NaCl. In view of this it is possible to carry out afirst step of cultivation at a lower sodium chloride concentration andto transfer the algae to a culture medium having a higher content ofsodium chloride or to increase the NaCl content of the medium.

The algae are quite large and heavy, especially when the cultivation isterminated at a high sodium chloride content and in view of this it iseasy to harvest the algae by sedimentation.

The glycerol and carotene can be recovered from the algae and thereremains a residue having a high protein content. The amino acidcomposition of the algae and of the algae meal remaining after solventextraction is as follows:

    ______________________________________                                        Amino Acids Analysis of                                                       Dunaliella bardawil                                                                                     Dry material after                                             Dried algae pellet                                                                           solvent extraction                                             (40% protein)  (70% protein)                                       Amino acid:                                                                              g/100 g protein                                                                              g/100 g protein                                     ______________________________________                                        Alanine    7.5            6.8                                                 Arginine   7.3            7.5                                                 Aspartic acid                                                                            10.6           10.6                                                Cysteine   1.2            1.3                                                 Glutamic acid                                                                            12.9           12.6                                                Glycine    5.7            5.9                                                 Histidine  1.8            1.7                                                 Isoleucine 4.2            4.0                                                 Leucine    11.0           11.1                                                Lysine     7.0            7.6                                                 Methoinine 2.3            1.7                                                 Phenylalanine                                                                            5.8            5.7                                                 Proline    3.3            2.8                                                 Serine     4.7            4.9                                                 Threonine  5.4            5.5                                                 Tryptophane                                                                              0.7            0.4                                                 Tyrosine   3.7            3.9                                                 Valine     5.8            5.7                                                 ______________________________________                                    

In the accompanying drawing, the FIGURE, magnified 4500×, showsDunaliella bardawil.

The characteristics of the algae in accordance with the presentinvention are summarized on the following table:

Nomenclature data:

Genus.--Dunaliella.

Species.--bardawil.

Authority.--Named by Dr. Ami Ben-Amotz and Prof. Mordhay Avron,Department of Biochemistry, Weizmann Institute of Science.

History and origin:

Isolated from.--Natural salt pond near Bardawil Lagoon, North Sinai,latitude 31°07', longitude 33°30'. This salt pond, known as LakeBardawil, is a cultivated pond, because nutrients are added to thispond.

Collected and isolated by.--Dr. A. Ben-Amotz.

Identified by.--Dr. A. Ben-Amotz and Prof. M. Avron.

Maintenance:

Method of preservation.--Agar culture. May be maintained also in liquidgrowth medium.

Temperature of preservation.--5° to 20° C.

Preservation suspending medium.--Liquid growth medium or 1.5-2% agaradded to growth medium on plates or slants.

Growth medium.--McLachlan, J. 1960, Can. J. Microbiol. 6, 367.Ben-Amotz, A., 1975, J. Phycol. 11, 50, with the addition of 1-4 M NaCl.

Growth conditions.--Bubbling 1-2% CO₂ in air, or addition of 10 mMNaHCO₃ to the growth medium in the presence of 20 mM TRIS, pH 7.5.

Special features and usage:

Anatomical part. Similar to that described by Butcher (Butcher, R. W.1959, Fish. Invest. Ser. 4,1) for D. salina except for size (length15-19 μm; diameter 10-14 μm; volume 200-1000 um³). Under definedconditions (see below), the alga contains up to 9% of its dry weight ascarotene isomers and up to 50% as glycerol.

Favorable conditions for high production of carotene

Light.--High light intensity (1500 f.c. up to full sunlight). When grownoutdoors in a thin layer (3-10 cm), the cells are bright orange, as aresult of massive accumulation of carotene (see table). When grownindoors with artificial illumination (200-400 f.c.), the cells graduallylose most of the orange color and become green. This transition isreversible.

NaCl.--High concentration of NaCl (above 3.0 M).

Nitrogen source.--Below 1 mM.

Diurnal cycle.--With the high light intensities optimal for caroteneproduction, growth is maximal only under a diurnal cycle; such a cyclecan be achieved by growth outdoors or by a 14 hours light--10 hours darkcycle indoors. Severe inhibition of growth occurs when the cells areexposed continuously (24 hours daily) to high light intensity.

    ______________________________________                                        Content of β -carotene, Glycerol, Chlorophyll and Protein                under favorable conditions:                                                                Growth outdoors,                                                                         Growth indoors,                                                    3 M NaCl   3 M NaCl                                              ______________________________________                                        β -carotene pg/cell                                                                     50-90         8-16                                             Glycerol pg/cell                                                                             300-400      200-275                                           Chlorophyll pg/cell                                                                          4-8          10-16                                             Carotene mg/g dry wt.                                                                        50-90        10-21                                             Glycerol mg/g dry wt.                                                                        300-400      270-370                                           Chlorophyll mg/g dry wt.                                                                      8-12        13-21                                             Protein mg/g dry wt.                                                                         300-400      300-400                                           ______________________________________                                    

What is claimed is:
 1. A new and distinct variety of alga of the genusDunaliella, named Dunaliella bardawil, substantially as herein shown anddescribed, characterized by the fact that when grown outdoors in a thinlayer the cells are bright orange, the cells containing up to 9% dryweight of carotene and up to 50% glycerol.